In the past 30 years, pathogenic microorganisms which are difficult to cultivate or can not be cultured have become the main source of contagious or infectious diseases. Prior art detection methods using markers such as number of proliferating bacterial colonies, colony purification separation, external morphology and physiological and biochemical identification as well as serological identification do not meet the fast, easy, high-specificity identification requirements of today because of their time-consuming, tedious steps and other shortcomings. Therefore, it is increasingly important to correctly identify these pathogenic microorganisms which are difficult to cultivate or cannot be cultured and to study these pathogenic bacteria nucleic acid structures and molecular characteristics at the molecular biology level thereby greatly enhancing the detection of such pathogenic bacteria. At present, highly sensitive, highly specific and rapid nucleic acid amplification technology can directly detect clinical specimens. However these techniques have been applied more widely in the infectious disease diagnoses and there is a trend to gradually replace traditional bacteria or virus cultivation.
Polymerase chain reaction (PCR) technology is the most widely used nucleic acid amplification technology currently employed. At present, the external nucleic acid amplification technology may be divided into two types. The first type is characterized by cycling temperatures in thermal insulation spots and includes PCR, ligase chain reaction (LCR), and transcription based amplification systems (TAS). The second type includes isothermal amplification systems such as the strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), transcription mediated amplification (TMA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase dependent amplification (HDA). These methods all share the common characteristic that the amplification reactions are carried out under a uniform temperature so thereby simplifying the instrumentation required for the amplification reaction.
Most of various nucleic acid amplification technologies are coupled to various detection methodologies such as electrophoresis, fluorescence, mass spectrometry or direct sequencing so as to detect target sequences that are amplified. The detection technologies often involve complicated operations, are costly and usually require large-scale equipment that must be operated by skilled professionals. Furthermore, these techniques are often not suitable for broad application and use in many rural third world hospitals. The present invention combines isothermal nucleic acid amplification technology and nucleic acid testing strip rapid detection for simple, rapid, low cost detection of pathogens as well as other nucleic acid containing organisms.
As previously described there are several methods of nucleic acid amplification. PCR is accomplished by providing oligonucleotide primers at both sides of the target sequence so as to enzymatically synthesize several target sequence DNA fragments. Each cycle of PCR includes the DNA double strand separation, primer renaturation and an extension reaction catalyzed by the DNA polymerase which makes newly synthesized DNA fragments that may again become the templates for next cycle of amplification thereby giving rise to the exponential amplification of the target sequence DNA. At present, PCR technology has been applied widely in various aspects in the biotechnology such as detection of genetic diseases, cancer diagnoses and prognosis, identification of bacteria, viruses and fungal infection; and paternity.
LCR is amplification based on the connection capability of the Taq ligase, which is able to detect point mutations in a target gene sequence. LCR may identify the specific point mutation more readily than PCR. If there are any point mutations in the target sequence, the primer may not be connected with the target sequence precisely. The nucleotide special structure near the mutation has been varied so as that LCR may not be carried out and the amplification products may also not be generated. At present, the method is mainly used in the research and detection on the point mutation, such as the diagnoses of polymorphisms and products of single base hereditary diseases, research on specific identification of microorganisms and point mutations in the cancer genes.
RCA is divided into two types: linear amplification and exponential amplification. The former may only be applicable for annular nucleic acid amplification, whose products are a large number of DNA single strands of the repeated sequences complementary to the annular DNA. This technique may be suitable for specific signal detection on micro-arrays or in the solid-phase forms. In exponential amplification, the amplification products may also act as the templates thereby increasing amplification products exponentially. This technique may also be used for the non-annular DNA amplification. The specificity of RCA is very high, thus it can be used for mutation detection and SNP identification. Its use can be integrated with the fluorescent real-time detection, thereby enabling broad use of the technique.
TMA is amplification of RNA or DNA utilizing reverse transcriptase and T7 RNA polymerase under the isothermal conditions. In TMA reverse transcription of the target is accomplished by the action of the reverse transcriptase under the guidance a primer. The H activity of RNA reverse transcriptase degrades the RNA in the DNA-RNA hybrid chain thereby permitting the synthesis of double stranded DNA which can further be transcribed into thousands of RNA sequences under the action of T7 RNA polymerase. These RNAs may also act as the templates for next cycle. The whole TMA reaction is one autocatalytic process. The specificity of this method is high as is sensitivity. The reaction conditions are simple and the amplification efficiency is high. TMA does not require special amplification instruments and the whole reaction may be carried out in 1 test tube thereby reducing environmental pollution.
Amplification relying on the nucleic acid sequences, called self-sustained sequence replication (3SR), is used primarily for RNA detection. The reaction depends on the reverse transcriptase, T7 RNA polymerase, nuclease H as well as two special primers. The 3′ end of the primer I, is complementary with the target sequence and its 5′ end contains T7 RNA polymerase promoter for cDNA synthesis. The sequences of the primer II are complementary with the 5′ end of the cDNA. During the reaction primer I is annealed to the RNA template to catalytically synthesize cDNA under the action of the reverse transcriptase. The RNA is then hydrolyzed by the nuclease H to form single-stranded DNA. Primer II is annealed to the 5′ end of the cDNA and a second DNA strand is synthesized thereby forming a double-stranded DNA containing the T7 RNA polymerase promoter. Reverse transcriptase is used to transcribe a new RNA strand that is identical to the sample RNA sequence. Each new RNA strand may also act as the template to synthesize cDNA. The process may be repeated to form more RNAs and cDNAs. The operation is simple; no special instrument is required; no temperature cycling is required. Amplification may not be effective if the double stranded DNA has no any promoter sequence so if the reaction specificity is increased greatly. This technology is suitable for detecting and quantitatively analyzing specific RNA and also applicable for amplifying double stranded DNA. Therefore, it may be applied widely in the clinic.
SDA relies on the use of restriction endonuclease and DNA polymerase. SDA requires single-strand DNA template preparation in which DNA fragment of interest is generated in which the two ends of the fragment include enzyme sites for SDA cycling. A primer containing the restriction endonuclease identification sites is combined with the single-stranded target molecules to form double-stranded DNA with semi-phosphorylation sulfation sites by the action of DNA polymerase which has no excision enzyme activity. The unprotected primer chain is cut by the restriction endonuclease; whereas, the modified target fragments remain intact and the DNA polymerase starts the extension at the notch location and replaces the downstream sequences so as to generate another DNA single strand whose notch may be opened by the restriction endonuclease. Such opening notches, polymerization and replacement procedures are recycled repeatedly thereby generating a large number of complementary strands of target molecules. SDA has the high sensitivity and can rapidly amplify single-stranded molecules; however, its application range is restricted because of the complexity of the target sequence preparation and detection method limitations.
LAMP is mainly made up of the Bst large-fragment DNA Polymerase and two pairs of special internal primers (FIP being made up of F1C and F2; BIP being made up of BIC and B2) and one pair of the external primer (F3 and B3). The F2 sequence of the FIP primer is coupled with the complementary sequence in the target DNA and the loop strand displacement reaction may be started. The F3 primer is complementary with the F3C area in the template to bring about and synthesize the double strands of the template DNA so as to crowd out the DNA single strand introduced by FIP. In the meantime, the BIP primer is combined with the crowded out single strand hybridization so as to open the formed annular structure. Then the B3 primer is coupled with the base at the BIP outer side to form the new complementary strand under the action of the polymerase. There are the complementary sequences in the both ends of the displaced single-stranded DNA so as that the self base coupling may occur to form the dumbbell DNA structure, which may act as the starting structure for LAMP reaction to recycle and extend and a large number of DNA sequences are generated repeatedly and alternatively to form the amplification products, which are stem-loop structure DNA with many loops and in the cauliflower shape. LAMP is highly specific and highly sensitive. Detection of pathogenic microorganisms using LAMP can be both qualitative and quantitative as there is a linear relation between the quantity of magnesium pyrophosphate precipitation generation and the quantity of DNA generated. LAMP has a simple experimental setup and the experiments are isothermal, thus only an ordinary water bath or other devices which can act as the stable heat source may be required. This method may have applicability for scientific research work as well as a routine detection tool.
TAS is primarily used for the amplification RNA. It utilizes reverse transcriptase, T7 RNA polymerase and nuclease H as well as two special primers. The 3′ end of the primer I is complementary with RNA for amplification and its 5′ end contains the promoter information of T7 RNA polymerase. The reverse transcriptase synthesizes cDNA by using primer I as the starting point. Primer II is complementary with the 3′ end of this cDNA and is used to synthesize the second strand of the cDNA. T7 RNA polymerase transcribes RNA which is the same as the RNA for amplification by taking the double stranded DNA as the template, which may be the template for the next round reaction. The TAS is with high amplification efficiency and its specificity is high; whereas, its cycling processes are complicated and the reverse transcriptase and T7-RNA polymerase may be added repeatedly; therefore, its further study will be carried out.
HDA is a method that simulates natural DNA duplication, as it uses unwindases to separate DNA strands. HDA may be carried out under the same temperature so as to optimize synthesis thereby reducing cost and power consumption that a thermal cycler would require. The present invention provides a method of isothermal amplification and nucleic acid detection in which one kind of strand displacement DNA polymerase (preferably Bst DNA polymerase) can be maintained for dozens of minutes at some certain constant temperature (about 62° C.), to carry out nucleic acid amplification reactions. Therefore, the methods of the present invention provide rapid nucleic acid amplification may be carried out quickly and effectively and in which only one simple thermostatic apparatus is required to carry out all amplification processes so as to greatly decrease the complexity of the reaction (No thermocycler required). The methods of the present invention also couple nucleic acid detection testing strip detection with cross priming amplification so as to develop one new rapid nucleic acid detection method which enable amplification and detection processes to be accomplished easily and simply. Template thermal denaturation, long-time temperature cycling, tedious electrophoresis and other processes are no longer required. The methods of the present invention are specific, simple and quick and may be applied broadly, with applications including diagnoses on the molecules directly related to human genetic diseases, detection of pathogenic microorganisms, estimation on the tumor or cancer diagnoses and prognosis and microorganism typing. Some isothermal amplification methods require initial denaturation of target DNA (Genomic DNA) at higher temperature before the isothermal reaction. CPA does not require initial thermal denaturation as it is truly an isothermal method.